Rapid and accurate differentiation of cell types within a heterogeneous solution is a challenging but important task for various applications in biological research and medicine. Flow cytometry is the gold standard in cell analysis and is regularly used for blood analysis (i.e., complete blood counts (CBC)) to determine general patient health, blood diseases, and HIV or AIDs disease progression through quantitative measurement and analysis of common cell populations in patients' blood samples. Flow cytometry, however, lacks sufficient throughput to analyze rare cells in blood or other dilute solutions in a reasonable time period because it is an inherently serial process. Moreover, these systems have high fixed costs, high operating costs (for sheath fluids, lysis buffers, etc.), and lack of portability, which makes them less than ideal for point-of-care or resource limited settings. Further, the complexity of today's flow cytometers requires trained personnel to operate, analyze, and maintain the systems, adding to operating costs.
Analysis of cells within a heterogeneous solution is a challenging but important task for various applications in biological research and medicine including general characterization of cellular protein content, identification of stem cells or tumor cells from dissociated tissue biopsies, and analysis of cell content in blood and other body fluids. Among cell analysis techniques, flow cytometry is most commonly used because of the quantitative data and significant throughputs (˜10,000 cells/s) achievable. Despite the current success of flow cytometry there is still interest in (1) further increasing throughput and, (2) bringing the availability of instruments to the point-of-care. Clinically, flow cytometry is often used for blood analysis (i.e., complete blood counts (CBC)) to determine general patient health, blood diseases, and HIV or AIDs disease progression. Today, a CBC test generally identifies subpopulations of white blood cells (WBCs), red blood cells (RBCs), platelets, hemoglobin, hermatocrit volume, and platelet volume, but does not identify a variety of rare cells (<10,000 cells/mL) that can also be present in blood and potentially clinically useful (e.g. hematopoietic stem cells, endothelial progenitor cells, and circulating tumor cells).
Statistically accurate identification of these cells is not possible in a reasonable time period using standard flow cytometry, especially in a background of 5×109 RBCs/mL, and even if RBCs are lysed to yield a background of ˜107 WBCs/ml. The limited throughputs possible in modern clinical bench top flow cytometers and hematology analyzers is due to the serial nature of the cell focusing and interrogation process. For example the gold standard Abbott Cell-Dyn Hematology Analyzer relies on optical scattering and fluorescence intensity measurements for identification and enumeration of blood components based on a sheath-flow hydrodynamic focusing platform. Upon analysis the system requires sequential chemical lysis of WBC and RBC populations to achieve higher specificity. The complexity in cell interrogation and the use of consumable reagents required for operation prevents parallelization for increased throughput required for rare cell detection purposes.
There is also considerable interest in decreasing the cost of flow cytometry and hematology instruments. These systems have high fixed costs (>$30,000), high operating costs (sheath fluids, lysis buffers), and lack of portability, which makes them less than ideal for point-of-care or resource limited settings. Further, the complexity of today's flow cytometers requires trained personnel to operate, analyze, and maintain the systems, further adding to operating costs. Thus a simpler system could reduce healthcare costs while also increasing patient access.
To address these challenges several efforts to miniaturize flow cytometry using microfluidic techniques have been previously explored such that costs are reduced, with the possibility of increased parallelization and throughput. In most cases, the macroscale mechanism of operation including “sheath flows” necessary to focus cells, and laser scatter and fluorescence are translated directly to the microscale with various miniaturization techniques employed to recapitulate the same macroscale performance. The apparent need for a robust, cost effective, flow cytometers have resulted in numerous advances toward miniaturizing flow cytometry to the microscale.
Flow cytometer systems have operated at rates as high as 17,000 cells/s for cell counts that rely on three separate fluid inputs to create the “sheath flow” necessary to focus cells to a single optically interrogated volume. Other flow cytometer systems utilize secondary flow around curving channels for sheath-based 3D hydrodynamic focusing to position cells to a single z-position. Each of these systems are able to achieve highly uniform cell positioning for WBC differentiation with a final throughput of 1,700 cell/s.
Another system includes a microflow cytometer in which the sample fluid was ensheathed and hydrodynamically focused into small interrogated volume (20×34×30 μm3) using a microstructured channel with chevron-shaped grooves. This system interrogated cells utilizing an embedded fiber-optic detection system allowing for miniaturization. Still other systems are shealthless microscale cytometer systems, which have the ability to differentiate and count blood components in the randomly dispersed flows. One such system uses laser light scattering techniques to achieve a 3-part WBC differentiation in addition to platelets, and RBC enumeration with a throughput of 1000 cells/s.
Still other systems achieve shealthless cell counts in microchannel flows by using differential impedance spectroscopy for detection, allowing for WBC enumeration and sub-type differentiation, achieving a throughput of 100 cells/s. Yet another system included parallelization of optical cytometry for the purpose of rare cell detection. This system successfully resulted in a 384-channel parallel microfluidic cytometer capable of handling large numbers of unique samples with a rare-cell sensitivity (20˜40 positive events in 1000 cells/μl). Although successful for the application of identifying cells expressing parathyroid hormone receptor, the system's low-throughput (1070 cells/s), large footprint (25×50 cm2), and complex confocal laser detection system, may inhibit its adoption as a flow cytometer substitute.
Further increases in throughput in these microscale systems have been challenging, mainly because the methods of cell focusing and optical interrogation are not trivially parallelized, and in the case of sheath flow, requires increases instrument bulk, minimizing the impact of the attempted miniaturization. In order to have a commercially viable impact on rare cell and point-of-care analysis, new systems will have to retain the accuracy but surpass the throughput of current benchtop systems while also being robust, cost efficient and easy to use. Thus there is a need for fundamentally new methods and systems to focus cells and collect data massively in parallel to meet these demands. There is also a need for a high throughput and cost effective flow cytometer.